We propose to analyze the senescence-associated decline in mammary epithelial growth, using a new culture system. Multicellar epithelial organoids will be isolated from mammary glands of young mice (6-12 weeks old), virgin and multiparous aged mice (26 months old), and outgrowths of hyperplastic alveolar nodule tissue (which is "immortal in vivo). Each organoid type will be embedded in a matrix of hydrated collagen gel and maintained in vitro for mone month. During this time, the organoids produce tubule-like outgrowths which project into the matrix and resemble ducts of the gland in vivo. Features of these tubules (tubule number, morphology (using light and elctron microscopy), growth rate) will be compared among the 4 populations of epithelial cells (young, old virgin, old multiparous, hyperplastic). These same parameters will be measured and compared in the presence of various mammary-active hormones including insulin, adrenal cortical steroids, ovarian steroids, prolactin, and thyroxine. The matrix cultures will then be used to test how changes in the mammary fat pad contribute to the senescence-related changes in growth of mouse mammary epithelium. Small fragments (200 Mu dia) of epithelium-free mammary fat pads (3 and 26 months old) will be co-suspended in collagen matrices along with epithelial organoids. Tubule parameters and hormone responsiveness of the 4 epithelial types in the presence of fat pad tissue will be measured and compared. Finally, any molecules which mediate the fat pad/epithelium interaction will be identified. Fragments of young and old fat pads will be incubated in serum-free medium for 24 hours. This medium will be assayed for ability to stimulate epithelial responses which resemble those elicited by intact adipose tissue. The fluid will be fractionated and active molecules identified. The results of this project should reveal why replication of mammary epithelium declines in an adipose tissue which supports replication in young animals. More generally, the studies will broaden our understanding not only of epithelial aging but also of changes in adipose tissue during aging and how these changes could impinge on other cell types of the body.